1. Field of the Invention
This invention relates to novel derivatives of lidocaine and related compounds (analyte) pertaining to immunoassays in liquid media such as biological fluids. Such derivatives include immunogens used to stimulate production of antibodies to the analyte of interest in host animals by conventional techniques. Also provided are labeled conjugates used as reagents, along with the antibodies, in particularly preferred immunoassays. Intermediates in the synthesis of the aforementioned immunogens and labeled conjugates are also provided.
Lidocaine [Merck Index, 9th ed., p. 5331 (1976)] is a local anesthetic of the formula: ##STR1## where Q is hydrogen and W.sup.1 and W.sup.2 are both ethyl, which also possesses antiarrhythmic properties, particularly against ventricular arrhythmias. It is widely used in the treatment of post-myocardial infarction patients where it is administered by bolus injection of 1 to 2 milligrams per kilogram, followed by constant infusion at a dose level of 20 to 50 micrograms per kilogram per minute. The toxic side effects hypotension, CNS depression, and convulsions appear to be avoidable if the blood levels do not exceed 5 micrograms per milliliter (ml). On long term constant infusions, 24 to 36 hours may be required to reach a steady state. Patients receiving such therapy need to be observed carefully and continuously for signs of lidocaine toxicity. Lidocaine and possibly lidocaine metabolite blood levels may have to be determined in order to treat arrhythmias effectively and fully understand the toxicity of the drug in a given patient. [Gianelly et al, New Engl. J. Med. 277: 1215 (1967); Winkle et al, Amer. J. Cardiology 36: 629 (1975)].
Lidocaine is metabolized in the liver by N-deethylation to produce monoethyl glycine xylidide [MEGX, formula (A) where Q=hydrogen, W.sup.1 =ethyl, W.sup.2 =hydrogen], and glycine xylidide [GX, formula (A) where Q=W.sup.1 =W.sup.2 =hydrogen]. In one study, the former metabolite was found to occur in blood in a concentration range of 0.31 to 2.6 micrograms per ml and was 80% as potent as an antiarrhythmic as lidocaine. The completely deethylated derivative (GX) was only one-tenth as potent as the parent drug [Burney et al, Amer. Heart J. 88: 765 (1974)]. In humans, elevated blood levels of MEGX have been linked with the central nervous system (CNS) side effects associated with lidocaine therapy. In rats the median convulsant dose of MEGX was determined to be 67 milligrams (mg) per kilograms as compared to 52 mg per kilogram for lidocaine itself, which suggests that the occurrence of seizures in humans treated with lidocaine may be partially due to the metabolite MEGX [Blumer et al, J. Pharm. Exp. Therap. 186: 31 (1973); Strong et al, Clin. Pharm. Therap. 14: 67 (1973)].
Tocainide, a primary amide analogue of lidocaine [formula (A) where Q is methyl and W.sup.1 =W.sup.2 =hydrogen], also possesses similar antiarrythmic properties and toxicity characteristics. Unlike lidocain, tocainide can be administered orally since it is not rapidly metabolized and eliminated in the first pass through the liver. Tocainide has a 10 hour half-life, is therapeutic at serum concentrations of 6 ng/ml, and has been used in combination with either quinidine or disopyramide. [Zipes and Troup, Amer. J. Cardiol. 41: 1005-1024 (1978)].
For optimum therapeutic management of patients medicated with the above-discussed drugs and metabolites, a rapid and specific analytical method is needed that is sensitive enough to measure plasma concentrations of the drug. This need has led to the development of a variety of analytical procedures to determine blood levels, particularly for lidocaine and MEGX. Some examples are mass fragmentography [Strong and Atkinson, Anal. Chem. 44: 2287 (1972)]; gas-liquid chromatography [Dusci and Hacket, Clin. Toxicol. 14: 587 (1979)]; high performance thin layer chromatography [Lee et al, J. Chromat. 158: 403 (1978)]; and enzyme-mediated immunoassay [Lebane et al, Clin. Chem. 25: 614 (1979)].
The preparation of antibodies to lidocaine and its analogs for use in immunoassays has been accomplished in the prior art by forming a particular immunogen conjugate of the drug and a conventional immunogenic carrier material and injecting such immunogen into the bloodstream of an appropriate host animal to stimulate antibody production. U.S. Pat. No. 4,069,105 describes such immunogen conjugates wherein the drug is coupled to the carrier through an imine linkage attached to one of the three unsubstituted positions on the lidocaine phenyl group. Specifically exemplified is attachment at the unsubstituted position which is para to the native amide side chain in the drug. Derivation of lidocaine at such para position through a different linkage for the purpose of forming labeled conjugates useful in a certain inhibitor-labeled immunoassay is proposed in U.S. Pat. No. 4,273,866.
The state-of-the-art of preparing antibodies to haptens such as drugs is represented by Weinryb et al, Drug Metabolism Reviews 10: 271 (1975); Playfair et al, Br. Med. Bull. 30: 24 (1974); Broughton et al, Clin. Chem. 22: 726 (1976); and Butler, J. Immunol. Meth. 7: 1 (1976) and Pharmacol. Rev. 29(2): 103 (1978).
Labeled conjugates, comprising the analyte or a derivative or other analog thereof, coupled to a labeling substance are variously described in the literature, e.g., U.S. Pat. Nos. 4,279,992; 4,182,856; 4,259,233; and 4,292,425 wherein the label is the fluorogenic enzyme substrate .beta.-galactosylumbelliferone, and U.S. Pat. No. 4,213,893, wherein the label is flavin adenine dinucleotide.